The use of in vitro technologies and high‐resolution/accurate‐mass LC‐MS to screen for metabolites of ‘designer’ steroids in the equine

Abstract

Detection of androgenic-anabolic steroid abuse in equine sports requires knowledge of the drug's metabolism in order to target appropriate metabolites, especially where urine is the matrix of choice. Studying 'designer' steroid metabolism is problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of toxicological data. In this study, the equine in vitro metabolism of eight steroids available for purchase on the Internet is reported; including androsta-1,4,6-triene-3,17-dione, 4-chloro,17α-methyl-androsta-1,4-diene-3,17β-diol, estra-4,9-diene-3,17-dione, 4-hydroxyandrostenedione, 20-hydroxyecdysone, 11-keto-androstenedione, 17α-methyldrostanolone, and tetrahydrogestrinone. In order to allow for retrospective analysis of sample testing data, the use of a high-resolution (HR) accurate-mass Thermo LTQ-Orbitrap liquid chromatography-mass spectrometry (LC-MS) instrument was employed for metabolite identification of underivatized sample extracts. The full scan LC-HRMS Orbitrap data were complimented by LC-HRMS/MS and gas-chromatography-mass spectrometry (GC-MS) experiments in order to provide fragmentation information and to ascertain whether GC-MS was capable of detecting any metabolite not detected by LC-HRMS. With the exception of 20-hydroxyecdysone, all compounds were found to be metabolized by equine liver S9 and/or microsomes. With the exception of 17α-methyldrostanolone, which produced metabolites that could only be detected by GC-MS, the metabolites of all other compounds could be identified using LC-HRMS, thus allowing retrospective analysis of previously acquired full-scan data resulting from routine equine drug testing screens. In summary, while in vitro techniques do not serve as a replacement for more definitive in vivo studies in all situations, their use does offer an alternative in situations where it would not be ethical to administer untested drugs to animals.

Metabolic study of androsta-1,4,6-triene-3,17-dione in horses using liquid chromatography/high resolution mass spectrometry

“…However, in the case of ATD, GC/MS was found to be not sensitive enough for the detection of both the in vitro and in vivo metabolites, most likely due to the extensive metabolism of ATD through the reductions of two ketone groups and three olefin groups. Liquid chromatography/high resolution mass spectrometry (LC/HRMS) has been proven to be useful and versatile for the identification and characterization of metabolites [13,[17][18]. The use of full-scan HRMS offers the advantage of the simultaneous detection of virtually an unlimited number of phase I metabolites, and also the direct detection of phase II metabolites such as glucuronide and sulfate conjugates.…”

“…In May 2014, aromatase inhibitors have been listed as one of the classes of hormone and metabolic modulators (Article 6E of the International Agreement of Breeding, Racing, and Wagering) which are prohibited to be administered to racehorses at any time in their career [11]. The metabolism of ATD has been reported in human [10,12], and the in vitro metabolic study of ATD in horses has been reported [13], but little is known about its biotransformation and elimination in horses. This paper describes the investigation of the in vitro and in vivo metabolism of ATD in horses with an objective to identify the most appropriate targets for detecting ATD administration.…”

Detection of new exemestane metabolites by liquid chromatography interfaced to electrospray-tandem mass spectrometry

“…All the features depicted above lead to the proposed metabolite 6-17␤-dihydroxyandrosta-1,4-dien-3-one. Another evidence is observed on the ESI product ion spectrum of 3-keto-1,4,6-triene steroids (17␣/␤-hydroxy-androsta-1,4,6-triene-3-one), its fragment ions are identical [19] with the fragment ions observed in ESI product ion spectrum of compound 6-17␤-dihydroxyandrosta-1,4-dien-3-one: m/z 159, m/z 171, m/z 173 and m/z 197, suggesting that the water neutral loss from C6 hydroxy group leads to a 3-keto-1,4,6-triene structure identical to the ones mentioned above (Fig. 6).…”

“…7). Its ESI fragmentation features are pretty similar to another 3-keto-1,4,6-triene steroids (androsta-1,4,6-triene-3,17-dione) [19]. Due to fragment ions associated with the compound mass and early RT the metabolites 6␣/␤-hydroxyandrosta-1,4-diene-3,17-dione (6␣/␤-hydroxyexemestane) were proposed.…”

Challenges in detecting substances for equine anti‐doping

“…The difficulty seen in the past of obtaining ethical approval for costly in vivo administrations has been overcome with attractive in vitro procedures due to satisfactory correlation factors. [57,58] The in vitro metabolism studies of desoxy-methyltestosterone (DMT), widely known as MADOL, and five of its structural analogues with different substituents at the 17α-position (R═H, ethyl, vinyl, ethynyl and H 3 -methyl) of typical steroid structure was studied in equine liver microsomes and the in vivo metabolism of one of the DMT analogues, desoxy-vinyltestosterone (DVT) using GC-MS in the electron ionization (EI) mode with full-scan acquisition was reported. [59] The detection and metabolic investigation of the novel designer steroid, 3-chloro-17α-methyl-5α-androstan-17β-ol, was performed highlighting its efficiency for doping.…”